Soluble laminin and arginine-glycine-aspartic acid containing peptides differentially regulate type IV collagenase messenger RNA, activation, and localization in testicular cell culture

Rat testicular cells in culture produce several metalloproteinases including type IV collagenases (Sang et al. Biol Reprod 1990; 43:946-955, 956-964). We have now investigated the regulation of testicular cell type IV collagenase and other metalloprotemases in vitro. Soluble laminin stimulated Sertoli cell type IV collagenase mRNA levels. However, three peptides corresponding to different domains of the laminin molecule (CSRAKQAASIKVASADR, FALRGDNP, CLQDGDVRV) did not influence type IV collagenase mENA levels. Zyniographic analysis of medium collected from these cultures revealed that neither soluble laminin nor any of the peptides influenced 72-Wa type IV collagenase protein levels. However, peptide FALRGDNP resulted in both, a selective increase in two higher molecular-weight metalloprotemnases (83 kDa and 110 Wa and in an activation of the 72-Wa rat type IV collagenase. Interleukin-1, phorbol ester, testosterone, and FSH did not affect collagenase activation, lmmunocytochemical studies demonstrated that the addition of soluble laminin resulted in a redistribution of type IV collagenase from intracellular vesicles to the cell-substrate region beneath the cells. Peptide FALRGDNP induced a change from a vesicular to peripheral plasma membrane type of staining pattern. Zymography of plasma membrane preparations demonstrated triton-soluble gelatinases of 76 Wa, 83 Wa, and 110 Wa and a triton-insoluble gelatinase of 225 Wa, These results indicate that testicular cell type IV collagenase mRNA levels, enzyme activation, and distribution are influenced by laminin and RGD-containing peptides.

Evaluation of a community-based intervention to promote physical activity in youth : lessons from active winners

Purpose To test the effects of a community-based physical activity intervention designed to increase physical activity and to conduct an extensive process evaluation of the intervention. Design Quasi-experimental. Setting Two rural communities in South Carolina. One community received the intervention, and the other served as the comparison. Subjects Public school students who were in fifth grade at the start of the study (558 at baseline) were eligible to participate. A total of 436 students participated over the course of the study. Intervention The intervention included after-school and summer physical activity programs and home, school, and community components designed to increase physical activity in youth. The intervention took place over an 18-month period. Measures. Students reported after-school physical activity at three data collection points (prior to, during, and following the intervention) using the Previous Day Physical Activity Recall (PDPAR). They also completed a questionnaire designed to measure hypothesized psychosocial and environmental determinants of physical activity behavior The process evaluation used meeting records, documentation of program activities, interviews, focus groups, and heart rate monitoring to evaluate the planning and implementation of the intervention. Results There were no significant differences in the physical activity variables and few significant differences in the psychosocial variables between the intervention and comparison groups. The process evaluation indicated that the after-school and summer physical activity component of the intervention was implemented as planned, but because of resource and time limitations, the home, school, and community components were not implemented as planned. Conclusions The intervention did not have a significant effect on physical activity in the target population of children in the intervention community. This outcome is similar to that reported in other studies of community-based physical activity intervention.

Involvement of focal adhesion kinase in inhibition of motility of human breast cancer cells by sphingosine 1-phosphate

Sphingosine 1-phosphate (SPP), a bioactive sphingolipid metabolite, inhibits chemoinvasiveness of the aggressive, estrogen-independent MDA-MB-231 human breast cancer cell line. As in many other cell types, SPP stimulated proliferation of MDA-MB-231 cells, albeit to a lesser extent. Treatment of MDA-MB-231 cells with SPP had no significant effect on their adhesiveness to Matrigel, and only high concentrations of SPP partially inhibited matrix metalloproteinase-2 activation induced by Con A. However, SPP at a concentration that strongly inhibited invasiveness also markedly reduced chemotactic motility. To investigate the molecular mechanisms by which SPP interferes with cell motility, we examined tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, which are important for organization of focal adhesions and cell motility. SPP rapidly increased tyrosine phosphorylation of FAK and paxillin and of the paxillin-associated protein Crk. Overexpression of FAK and kinase-defective FAK in MDA-MB-231 cells resulted in a slight increase in motility without affecting the inhibitory effect of SPP, whereas expression of FAK with a mutation of the major autophosphorylation site (F397) abolished the inhibitory effect of SPP on cell motility. In contrast, the phosphoinositide 3'-kinase inhibitor, wortmannin, inhibited chemotactic motility in both vector and FAK-F397- transfected cells. Our results suggest that autophosphorylation of FAK on Y397 may play an important role in SPP signaling leading to decreased cell motility.

Determinants of physical activity in Singaporean adolescents

Backgroun An understanding of the factors that influence physical activity behavior is an important prerequisite for the design and implementation of physical activity interventions in adolescents. To date, no studies have investigated the factors that influence physical activity participation in Singaporean adolescents. Purpose he purpose of this study was to identify the psychosocial and environmental factors that influence physical activity in a representative sample of Singaporean adolescents (N=1,814, 919 boys, 895 girls, mean age 14.4 +/- 1.1 years). Methods Participants completed the Three-Day Physical Activity Recall and a questionnaire measuring hypothesized psychosocial and environmental correlates of physical activity. Results Hierarchical regression revealed self-efficacy, enjoyment of physical activity, parental support, and participation in sport teams to be significant correlates of physical activity. Conclusion Interventions promoting physical activity in Singaporean adolescents should aim to increase self-efficacy perceptions by offering enjoyable, developmentally appropriate physical activity options that promote mastery and adopt policies that increase parental support and awareness of community physical activity programs.

Correlates of physical activity behavior in rural youth

The purpose of this study was to identify correlates of physical activity behavior in a sample of rural, predominantly African American youth. Three hundred sixty-one fifth-grade students from two rural counties in South Carolina (69% African American, median age = II years) completed a questionnaire designed to measure beliefs and social influences regarding physical activity, physical activity self-efficacy, perceived physical activity habits of family members and friends, and access to exercise and fitness equipment at home. After school physical activity and television watching were assessed using the Previous Day Physical Activity Recall (PDPAR). Students were classified as physically active according to a moderate physical activity standard: two or more 30-min blocks at an intensity of 3 METs (metabolic equivalents) or greater, and a vigorous physical activity standard: one or more 30-min blocks at an intensity of 6 METs or greater According to the moderate physical activity standard, 34.9% of students were classified as low-active. Multivariate analysis revealed age, gender television watching, and exercise equipment at home to be significant correlates of low activity status. According to the vigorous physical activity standard, 32.1 % of the students were classified as low-active. Multivariate analysis revealed age, gender television watching, and self-efficacy with respect to seeking support for physical activity to be significant correlates of low activity status. In summary, gender and the amount of television watching were found to be the most important correlates of physical activity in rural, predominantly African American youth.

Psychosocial correlates of physical activity in white and African-American girls

Purpose To evaluate the relative utility of the Theory of Reasoned Action (TRA) and the Theory of Planned Behavior (TPB) in explaining intentions and physical activity behavior in white and African-American eighth-grade girls. Methods One-thousand-thirty white and 1114 African-American eighth-grade girls (mean age 13.6 ± 0.7 years) from 31 middle schools in South Carolina completed a 3-day physical activity recall and a questionnaire assessing attitudes, subjective norms, perceived behavioral control, self-efficacy, and intentions related to regular participation in moderate-to-vigorous physical activity (MVPA). Results Among Whites, 17% of the variance in intentions was contributed by subjective norms and attitude, with intentions accounting for 8% of the variance in MVPA. The addition of perceived behavioral control and self-efficacy to the TRA significantly improved the prediction of intentions and MVPA accounting for 40% and 10% of the variance, respectively. Among African-Americans, subjective norms and attitude accounted for 13% of the variance in intentions, with intentions accounting for only 3% of the variance in MVPA. The addition of perceived behavioral control and self-efficacy to the TRA significantly improved the prediction of intentions and MVPA accounting for 28% and 5% of the variance, respectively. Conclusions The results provided limited empirical support for the TPB among white adolescent girls; however, our findings suggest that the planned behavior framework has limited utility among African-American adolescent girls. The relatively weak link between intentions and MVPA observed in both population groups suggest that constructs external to the TPB may be more important mediators of physical activity behavior in adolescent girls.

Physical activity correlates in adolescent girls who differ by weight status

Objective This study compared correlates of physical activity (PA) among African-American and white girls of different weight groups to guide future interventions. Research Methods and Procedures Participants were 1015 girls (mean age, 14.6 years; 45% African-American) from 12 high schools in South Carolina who served as control subjects for a school-based intervention. Post-intervention measures obtained at the end of ninth grade were used. PA was measured using the Three-Day PA Recall, and a questionnaire measured social-cognitive and environmental variables thought to mediate PA. Height and weight were measured, and BMI was calculated. Girls were stratified by race and categorized into three groups, based on BMI percentiles for girls from CDC growth charts: normal (BMI < 85th percentile), at risk (BMI, 85th to 94th percentile), and overweight (BMI ≥ 95th percentile). Girls were further divided into active and low-active groups, based on a vigorous PA standard (average of one or more 30-minute blocks per day per 3-day period). Mixed-model ANOVA was used to compare factors among groups, treating school as a random effect Results None of the social-cognitive or environmental variables differed by weight status for African-American or white girls. Perceived behavioral control and sports team participation were significantly higher in girls who were more active, regardless of weight or race group. In general, social-cognitive variables seem to be more related to activity in white girls, whereas environmental factors seem more related to activity in African-American girls. Discussion PA interventions should be tailored to the unique needs of girls based on PA levels and race, rather than on weight status alone.

Physical activity and physical fitness in African-American girls with and without obesity

Lack of physical activity and low levels of physical fitness are thought to be contributing factors to the high prevalence of obesity in African-American girls, To examine this hypothesis, we compared habitual physical activity and physical fitness in 54 African-American girls with obesity and 96 African-American girls without obesity residing in rural South Carolina, Participation in vigorous (greater than or equal to 6 METs) (VPA) or moderate and vigorous physical activity (greater than or equal to 4 METs) (MVPA) was assessed on three consecutive days using the Previous Day Physical Activity Recall, Cardiorespiratory fitness was assessed using the PWC 170 cycle ergometer test, Upper body strength was determined at two sites via isometric cable tensiometer tests, Relative to their counterparts without obesity, girls with obesity reported significantly fewer 30-minute blocks of VPA (0.90 +/- 0.14 vs. 1.3 +/- 0.14) and MVPA (1.2 +/- 0.18 vs. 1.7 +/- 0.16) (p<0.01), Within the entire sample, VPA and MVPA were inversely associated with body mass index (r=-0.17 and r=-0.19) and triceps skinfold thickness (r=-0.19 and r=-0.22) (p<0.05), In the PWC 170 test and isometric strength tests, girls with obesity demonstrated absolute scores that were similar to, or greater than, those of girls without obesity; however, when scores were expressed relative to bodyweight, girls with obesity demonstrated significantly lower values (p<0.05). The results support the hypothesis that lack of physical activity and low physical fitness are important contributing factors in the development and/or maintenance of obesity in African-American girls.

Physical activities and sedentary pursuits in African American and Caucasian girls

The purposes of this study were to describe and compare the specific physical activity choices and sedentary pursuits of African American and Caucasian American girls. Participants were 1,124 African American and 1,068 Caucasian American eighth grade students from 31 middle schools. The 3-Day Physical Activity Recall (3DPAR) was used to measure participation in physical activities and sedentary pursuits. The most frequently reported physical activities were walking, basketball, jogging or running, bicycling, and social dancing. Differences between groups were found in 11 physical activities and 3 sedentary pursuits. Participation rates were higher in African American girls (p<.001)for social dancing, basketball, watching television, and church attendance but lower in calisthenics, ballet and other dance, jogging or running, rollerblading, soccer, softball or baseball, using an exercise machine, swimming, and homework. Cultural differences of groups should be considered when planning interventions to promote physical activity.

Regulation of breast cancer cells by hormones and growth factors : effects on proliferation and basement membrane invasiveness

The current understanding of the regulation of breast cancer cell proliferation and invasiveness by hormones and growth factors is reviewed. It has been shown that polypeptide growth factors are involved in hormone-independent breast cancer, and are sometimes oestrogen-regulated in hormone-responsive models. Basement-membrane invasiveness, relating to the metastatic potential of these cells, is also stimulated by oestrogen in hormone-dependent models, elevated in hormone-independent models, and is growth factor sensitive. Further understanding of the differential effects of growth factors on breast cancer cell proliferation and invasiveness should facilitate better therapeutic exploitation of regulation at this level.

Heregulin regulates the actin cytoskeleton and promotes invasive properties in breast cancer cell lines

The metastatic process requires changes in tumor cell adhesion properties, cell motility and remodeling of the extracellular matrix. The erbB2 proto-oncogene is overexpressed in approximately 30% of breast cancers and is a major prognostic parameter when present in invasive disease. A ligand for the erbB2 receptor has not yet been identified but it can be activated by heterodimerization with heregulin (HRG)-stimulated erbB3 and erbB4 receptors. The HRGs are a family of polypeptide growth factors that have been shown to play a role in embryogenesis, tumor formation, growth and differentiation of breast cancer cells. The erbB3 and erbB4 receptors are involved in transregulation of erbB2 signaling. The work presented here suggests biological roles for HRG including regulation of the actin cytoskeleton and induction of motility and invasion in breast cancer cells. HRG-expressing breast cancer cell lines are characterized by low erbB receptor levels and a high invasive and metastatic index, while those which overexpress erbB2 demonstrate minimal invasive potential in vitro and are non-tumorigenic in vivo. Treatment of the highly tumorigenic and metastatic HRG-expressing breast cancer cell line MDA-MB-231 with an HRG-neutralizing antibody significantly inhibited proliferation in culture and motility in the Boyden chamber assay. Addition of exogenous HRG to non-invasive erbB2 overexpressing cells (SKBr-3) at low concentrations induced formation of pseudopodia, enhanced phagocytic activity and increased chemomigration and invasion in the Boyden chamber assay. The specificity of the chemomigration response to HRG is demonstrated by inhibition with the anti-HRG neutralizing antibody. These results suggest that either HRG can act as an autocrine or paracrine ligand to promote the invasive behavior of breast cancer cells in vitro or thus may enhance the metastatic process in vivo.

Secreted products and extracellular matrix from testicular peritubular myoid cells influence androgen-binding protein secretion by Sertoli cells in culture

Metabolic cooperation mediated by secreted factors between Sertoli cells and peritubular myoid cells has been well documented. We have confirmed that factors secreted by peritubular myoid cells modulate androgen-binding protein (ABP) secretion by Sertoli cells and shown further that this can also be achieved with peritubular myoid cell extracellular matrix (ECM). While peritubular myoid cell ECM potentiated the stimulatory effect of dibutyryl cyclic AMP on Sertoli cell ABP secretion, secreted factors did not, suggesting that the two components influence Sertoli cells through distinct mechanisms. We also tested other factors and other cell lines for effects on ABP production by Sertoli cells. The addition of human plasma fibronectin or conditioned medium from the basement membrane-producing Englebreth-Holm- Swarm sarcoma also stimulated ABP secretion by Sertoli cells. Cocultures of epithelial Sertoli cells with the cells of mesenchymal origin, such as testicular peritubular myoid cells, embryonic skin fibroblasts, and bladder smooth muscle cells, significantly stimulated ABP secretion by Sertoli cells, but co-culture with the epithelial-derived Martin-Darby canine kidney cell line had no effect on Sertoli cell-secreted ABP levels. Our data further define the epithelial-mesenchymal cell interaction that exists between Sertoli cells and peritubular myoid cells in the mammalian testis.

Type I collagen abrogates the clathrin-mediated internalization of membrane type 1 matrix metalloproteinase (MT1-MMP) via the MT1-MMP hemopexin domain

Type I collagen (Col I)-stimulated matrix metalloproteinase-2 (MMP-2) activation via membrane type 1 MMP (MT1-MMP) involves both a transcriptional increase in MT1-MMP expression and a nontranscriptional response mediated by preexisting MT1-MMP. In order to identify which MT1-MMP domains were required for the nontranscriptional response, MCF-7 cells that lack endogenous MT1-MMP were transfected with either wild type or domain mutant MT1-MMP constructs. We observed that mutant constructs lacking the MT1-MMP cytoplasmic tail were able to activate MMP-2 in response to Col I but not a construct lacking the MT1-MMP hemopexin domain. Col I did not alter total MT1-MMP protein levels; nor did it appear to directly induce MT1-MMP oligomerization. Col I did, however, redistribute preexisting MT1-MMP to the cell periphery compared with unstimulated cells that displayed amore diffuse staining pattern. In addition, Col I blocked the internalization of MT1-MMP in a dynamin-dependent manner via clathrin-coated pit-mediated endocytosis. This mechanism of impaired internalization is different from that reported for concanavalin A, since it is not mediated by the cytoplasmic tail of MT1-MMP but rather by the hemopexin domain. In summary, upon Col I binding to its cell surface receptor, MT1-MMP internalization via clathrin-coated pit-mediated endocytosis is impaired through interactions with the hemopexin domain, thereby regulating its function and ability to activate MMP-2.

Implication of collagen type I-induced membrane-type 1-matrix metalloproteinase expression and matrix metalloproteinase-2 activation in the metastatic progression of breast carcinoma

We have previously demonstrated that fibroblasts and invasive human breast carcinoma (HBC) cells specifically activate matrix metalloproteinase- 2 (MMP-2) when cultured on 3-dimensional gels of type I collagen but not a range of other substrates. We show here the constitutive expression of membrane-type 1 (MT1)-MMP in both fibroblasts, and invasive HBC cell lines, that have fibroblastic attributes presumably acquired through an epithelial- to-mesenchymal transition (EMT). Treatment with collagen type I increased the steady-state MT1-MMP mRNA levels in these cells but did not induce either MT1-MMP expression or MMP-2 activation in noninvasive breast carcinoma cell lines, which retain epithelial features. Basal MT3-MMP mRNA expression had a pattern similar to that of MT1-MMP but was not up-regulated by collagen. MT4- MMP mRNA was seen in both invasive and noninvasive HBC cell lines and was also not collagen-regulated, and MT2-MMP mRNA was not detected in any of the HBC cell lines tested. These data support a role for MT1-MMP in the collagen- induced MMP-2-activation seen in these cells. In situ hybridization analysis of archival breast cancer specimens revealed a close parallel in expression of both collagen type I and MT1-MMP mRNA in peritumoral fibroblasts, which was correlated with aggressiveness of the lesion. Relatively high levels of expression of both mRNA species were seen in fibroblasts close to invasive tumor nests and, although only focally, in certain areas close to preinvasive tumors. These foci may represent hot spots for local degradation and invasive progression. Collectively, these results implicate MT1-MMP in collagen- stimulated MMP-2 activation and suggest that this mechanism may be employed in vivo by both tumor-associated fibroblasts and EMT-derived carcinoma cells to facilitate increased invasion and/or metastasis.

The type I collagen induction of MT1-MMP-mediated MMP-2 activation is repressed by αVβ3 integrin in human breast cancer cells

The influence of αVβ3 integrin on MT1-MMP functionality was studied in human breast cancer cells of differing β3 integrin status. Overexpression of β3 integrin caused increased cell surface expression of αV integrin and increased cellular adhesion to extracellular matrix (ECM) substrates in BT-549, MDA-MB-231 and MCF-7 cells. β3 integrin expression also enhanced the migration of breast cancer cells on ECM substrates and enhanced collagen gel contraction. In vivo, αVβ3 cooperated with MT1-MMP to increase the growth of MCF-7 cells after orthotopic inoculation in immunocompromised mice, but had no influence on in vitro proliferation. Despite these stimulatory effects, overexpression of β3 integrin suppressed the type I collagen (Col I) induced MMP-2 activation in all breast cancer cell lines analyzed. This was also evident in extracts from the MCF-7 tumors in vivo, where MMP-2 activation was stimulated by MT1-MMP transfection, but attenuated with β3 integrin expression. Although our studies confirm important biological effects of αVβ3 integrin on enhancing cell adhesion and migration, ECM remodeling and tumor growth, β3 integrin caused reduced MMP-2 activation in response to Col I in vitro, which appears to be physiologically relevant, as it was also seen in tumor xenografts in vivo. The reduction of MMP-2 activation (and thus MT1-MMP activity) by αVβ3 in response to Col I may be important in scenarios where cells which are activated for matrix degradation need to preserve some pericellular collagen, perhaps as a substrate for cell adhesion and migration, thus maintaining a balanced level of proteolysis required for efficient tumor growth.